Correct control of medium addition rate to fermentation processes where accumulation of metabolites is to be prevented is a primary objective. Some microorganisms produce undesirable metabolites when fed at too high a medium addition rate. Examples are Bakers' yeast and Escherichia coli (De Deken, 1966; Doelle, 1981). Bakers' yeast will produce fermentation products such as ethanol and acetate when too much sugar is added (Fiechter et al, 1981). During the production of Bakers' yeast this will cause a loss of cell and product yield (Fiechter et al, 1981). The bacterium E. coli will produce acids such as acetic acid at sugar excess (Doelle, 1981). Also when microorganisms are used for the production of heterologous products the formation of these metabolites is undesirable, especially when these have a toxic or inhibitory effect. Acetate, ethanol and organic acids in general can be toxic to cell metabolism (Moon, 1983; Pampulha & Loureiro-Dias, 1989). This will become particularly apparent when growing mutant strains, which are often less robust than the wild-type strain. Therefore, good control of the feed addition rate to a fed-batch or continuous fermentation process is desirable.
Many ways of on-line computer control are possible. For example CO.sub.2 evolution rates and O.sub.2 consumption rates are often analysed on-line to calculate the so-called Respiratory Quotient (RQ) (Wang et al, 1977). The RQ is the CO.sub.2 evolution rate divided by the O.sub.2 consumption rate. Under sugar-limited conditions the RQ will be approximately 1.multidot.0 to 1.multidot.1, the exact value depending on the strain. However, when a culture of Bakers' yeast is fed at too high a sugar addition rate ethanol will be produced and the RQ values in that case will then be significantly higher than 1.multidot.1 (Wang et al, 1977; Fiechter et al, 1981). This then can be used to change the feed rate such that the RQ decreases (Wang et al, 1977).
EP 283 726 (Hitachi) and Turner et al (1994) disclose the control of fermentations by monitoring acetate levels, but the control was achieved by sampling the medium and using HPLC or similar discontinuous methods. HPLC has also been used to measure glucose levels in order to control acetate accumulation (Sakamoto et al, 1994).
The problem which is solved by the present invention is to provide an alternative and improved method of controlling such fermentations.
One aspect of the present invention provides a process of culturing a microorganism in a culture medium in which process the addition of feed medium is controlled by using the production of a by-product as a measure of the culture conditions, characterised in that the by-product is an electrically charged metabolite produced by the microorganism, and in that the production of the metabolite is monitored by measuring the conductance of the culture medium.
The evolution of electrically charged metabolites has not been used previously to control the addition of feed medium. RQ, for example, is 1 (one) when acetate is produced in a sugar fermentation, so RQ measurement is not useful, as this RQ value is near that obtained during sugar-limited growth. Electrical conductivity has been used to measure the formation of relatively large amounts of desired organic acids such as lactate in yogurt cultures and other lactobacillus fermentations (Latrille et al, 1992; Belfares et al, 1993), acetic acid production (SU-A-1 495 367) and for the control of salt content of fermentation cultures (Soyez et al, 1983). In the latter case, inorganic salts were added to the medium, and the technique simply measured those artificially added salts in order to maintain a desired salt concentration. Conductivity has also been used to measure cell density (JP-A-2 109 973). Conductivity has not been used to prevent and overcome the accumulation of undesirable acids such as acetate, of which even small amounts are indicative of the fermentation going awry. We have discovered that where the formation of organic acids such as acetate is undesirable, an increase in electrical conductivity can be measured on-line and used for a feed-back system to control the feed rate in a similar way as the RQ can be used. In this invention it is shown that increases in an on-line electrical conductivity signal during a fermentation process are sufficiently indicative of the formation of undesirable acids to be used to correct the feed addition rate in order to prevent and overcome accumulation of these acids. Hence, although for many years it has been known to measure (in an off-line biochemical assay) the production of acetate in order to see whether the fermentation control based on other parameters (eg CO.sub.2 evolution) is working satisfactorily (see EP 315 944, 1989), nobody had measured acetate evolution electrically to control fermentation.
Obviously, the microorganism and the fermentation medium should be such that an electrically charged metabolite is potentially produced and the fermentation should be one in which controlling the addition of feed medium is desirable. Equally, the fermentation should not be one in which an electrically charged product is desired, for example a lactic acid fermentation. Microorganisms for which the present invention is useful include bacteria such as E. coli or Bacilli and fungi such as yeasts, for example Saccharomyces spp., especially S. cerevisiae, or filamentous fungi. However, the invention is in principle applicable also to the culturing of protozoa, plant cells and animal cells, for example insect cells or mammalian cells such as CHO (Chinese Hamster Ovary) cells.
The metabolite is typically an organic acid such as acetate, pyruvate, lactate or a citric acid cycle intermediate such as citrate, isocitrate, .alpha.-ketoglutarate, succinate, fumarate, malate or oxaloacetate.
The microorganism may be cultured to produce either biomass, a desired metabolite or a polypeptide which is native or heterologous to the microorganism. Hence, for example, the microorganism may be a yeast which contains and expresses a polynucleotide encoding human albumin. Advantageously, the polypeptide is secreted from the yeast into the surrounding medium and recovered therefrom.
The measurement of the conductivity is very sensitive and can detect acid concentrations as low as 1 mM. This means that it is a useful alternative, or addition, to the generally accepted use of on-line RQ measurements.
The control may be achieved by use of a probe, capable of measuring conductivity, inserted in a fermenter and linking the signal to an on-line computer. A conductivity probe can be very simply inserted in a standard pH probe port. A computer algorithm can then calculate the change in conductivity or conductance over a chosen time period. If the change in conductivity is greater than a chosen limit, a reduction in the feed medium addition rate will automatically be applied by the computer algorithm. This will then promote a co-consumption of the feed substrate and the accumulated metabolites present, and prevent further production thereof. The choice of time period and conductivity change limit will be dependant on the exact nature of the fermentation process.